Fig 1.
CA1 pyramidal neurons loaded with Oregon Green 488 BAPTA-1.
Acute hippocampal slices (400 μm thick) were prepared from 4- to 8-week-old Wister rats and a slice was placed in a submerged type recording chamber mounted on the stage of an upright microscope equipped with a confocal microscopy system. (A), Top panel: The tip of a glass pipette filled with buffer solution containing 500 μM Oregon Green 488 BAPTA-1 AM was positioned in the str. radiatum 50 μm below the surface and 90 μm from the cell layer, and then a pressure of 70 kPa was applied for 40–50 minutes to eject the dye-containing solution from the pipette. After loading, the pipette was removed. Bottom panel: A confocal fluorescence image at low magnification (x16 objective lens). Note that many dendrites and non-pyramidal neurons are visible in the distal part of the str. radiatum and str. lacnosum-moleculare. (B)-(D) Confocal fluorescence images at a higher magnification (x40 objective lens). The soma and basal dendrites (B), tuft of the apical dendrites (C) and the shaft of the apical dendrites (D) of multiple neurons are loaded with the dye.
Fig 2.
Sinusoidal electrical field stimulation.
(A) Electric current was applied with a pair of Ag/AgCl electrodes placed 5 mm apart in parallel on the bottom of a submerge type recording chamber. A piece of hippocampal slice preparation was placed between the electrodes. A sinusoidal wave form was generated using in-house developed software run on a PC with a 12-bit digital-to-analog converter. The voltage output was electrically isolated and converted to current using a voltage-to-current converter. (B) Examples of extracellular field potential recorded from the CA1 area of a slice preparation. Field potential was recorded at 360 μm interval across the CA1 region from the basal to apical dendrite during the passage of 6 Hz sinusoidal current with a glass pipette inserted into the tissue of the slice. The potential was measured with respect to a reference electrode placed at the middle of str. radiatum throughout the recording. (C) Peak values of extracellular field potential at the top and bottom of the stimulation plotted against the location of the electrode. With our setup arrangement, the field intensity was uniform along the somato-dendritic axis and was independent of whether the recording electrode was inserted into the slice or was placed outside of the slice. The peak intensity of the sinusoidal electric field stimulation was 27.8 mV/mm in this particular case. Unless specified otherwise, the frequency of the sinusoidal wave was 4 Hz (more precisely 3.79 Hz or 3.95 Hz).
Fig 3.
Typical Ca fluorescence signals recorded from apical dendrites.
(A) Three types of spontaneous Ca-transient seen in ACSF/4-AP containing 3.75 mM K+ ion. Left panel: The field of view. The center of the image field was 300 μm from the border between the cell layer and the str. pyramidale. Six ROIs were selected by drawing lines around brightly stained neurites. Right panel: Time-course of the fluorescence intensity of the ROIs shown in the left panel. A round shaped cell (ROI #1) showed slow-transients, while dendritic branches (ROI #2–6) showed fast-transients. The transients marked with blue bars are local-fast-Ca-transients, while those marked with red bars are global-fast-Ca-transients. (B) Fast-Ca-transients in ACSF with different composition. The recordings were made from the same field with a slightly altered focus. In the middle and right panels, the ROIs were determined using the NMF-based algorithm and the data for all ROIs reported by the NMF-based algorithm are shown. Left panels: Responses in standard ACSF containing 2.5 mM K+ ion. No Ca-transient was detected during this session. Hence the ROIs were selected by drawing lines around brightly stained neurites by eye. Scale bar (ΔF/F): 10%. Center panels: In ACSF/4-AP containing 4.5 mM K+ ion. During the 30 sec recording session, 9 ROIs showed fast-Ca-transients. Scale bar (ΔF/F): 10%. Right panels: In ACSF/4-AP containing 5.5 mM K+ ion. Fast-Ca-transients were detected from 32 ROIs. Scale bar (ΔF/F): 20%.
Fig 4.
Spontaneous fast-Ca-transients recorded from different parts of a pyramidal neuron.
(A) Distal part of the apical dendrites. The center of the image field was 300 μm from the border between the cell layer and the str. pyramidale. The cASCF contained 7.5 mM K+ ion. (B) Basal dendrites. The cACSF contained 6.5 mM K+ ion. (C) Soma. The cACSF contained 3.0 mM K+ ion. All the ROIs in panel (A)-(C) were determined using the NMF-based algorithm. Frame rate 16.4 msec. Time course of the fluorescent intensity recorded at the ROIs shown on the left panels with different colors are shown on the right panels using the same colors. Scale bar (ΔF/F): 20%. (D) Comparison of the decay time constant (τ) of the fast-transients from different locations. Frame interval: 16.4 msec. Data for all fast-Ca-transients from a representative recording trial recorded at respective locations of each slice preparation were digitally averaged. For respective locations, 4 ROIs were randomly selected from each of the trials, and the decay time constants were obtained from the averaged traces. The decay phase of the traces was fitted to a double exponential function, and the values for the shortest time constants are shown.
Fig 5.
Pharmacological characterization of spontaneous fast-Ca-transients.
(A)—(C) Fast-Ca-transients recorded from the distal apical dendrites of the same preparation. (A) In cACSF with 3.5 mM K+ ion. (B), In cACSF containing 10 μM CNQX and 50 μM DL-APV (C). In the presence of 1 μM TTX added to the solution used in (B). Scale bar (ΔF/F): 20%. The recordings in (B) and (C) were made 15 minutes after switching the solution. (D) ROIs for panels (A)—(C). To allow comparison, the same ROIs were used throughout. The center of the image field was 450 μm from the border between the cell layer and the str. pyramidale. (E) Frequency of the fast-Ca-transients in 24 ROIs in the absence or presence of CNQX and DL-APV; the red line shows the mean values. There was slight increase in frequency in the presence of CNQX and DL-APV (153 ± 256%), but the change was not significant (Wilcoxon signed-rank test P>0.05, 4 slices, 24 ROIs). (F) Interval histogram of fast-Ca-transients in 24 ROIs in the absence or presence of CNQX and DL-APV constructed using the same set of data used for panel E; the blue bars show number of transient-intervals in cACSF, red bars show number of transient-intervals in the presence of CNQX and DL-APV (bin width: 2 sec). Inset shows the cumulative fraction of transient-intervals.
Fig 6.
Analysis of the impact of sinusoidal extracellular electric fields on fast-Ca-transients.
(A) Arrangement of the electrodes for field stimulation. Electric current was applied between a pair of Ag/AgCl electrodes to generate electric field. The intensity of the field was monitored by measuring bath potential. (B) ROIs for the data shown in (C). The center of the image field was 350 μm from the border between the str. pyramidale and str. radiatum. (C) Representative data showing fast-Ca-transients during sinusoidal field stimulation. After recording Ca signals for 20 sec in ACSF/4-AP containing 7.5 mM K+ ion, field stimulation (2.7 mV/mm, 2 Hz (1.88 Hz), 20 cycles)was turned on for 20 sec, then off for further 20 sec while recording the signals. Scale bar (ΔF/F): 40%. The onsets of fast-Ca-transients are marked with bars of the same color as the ROIs shown in (B). (D) Direction of the stimulating current and the phase. (E) Expanded traces of the signals near the onset of stimulation. (F) Frequency of Ca-transients during the periods in which the stimulation was turned on (colored bars, mean frequency 0.42 ± 0.22 Hz) or turned off (black, mean frequency: 0.29 ± 0.18 Hz) for ROIs indicated by the same colors in (B). (G) Spiral plot of the data shown in (C). The right panel shows data during the period in which the field was applied, and the left panel for the period with no field. The same angular velocity was used for both panels. Note that the colored bars are mainly located within the range of 60–240° during field stimulation.
Fig 7.
Entrainment of fast-Ca-transients at different field intensities.
(A,B,C) 4 Hz sinusoidal electric field of three different intensities was applied to the same slice preparation in ACSF/4-AP containing 6.5 mM K+ ion. Image recording was made from the distal apical dendrites with a frame interval of 16.4 msec. Analysis of data obtained in experiments with a field intensity of 12.5 mV/mm (A), 3.8 mV/mm (B), or 1.3 mV/mm (C) are shown. Top panels (a): Spiral plots. The right panels show data recorded during the period in which the field stimulation was applied (Field ON) and the left panels for the period with no applied field (Field OFF). Note that the fast-Ca-transients were entrained to the applied field even at the field intensity of 1.3 mV/mm. Upper middle panels (b): Phase distribution histograms for the data shown in (a). Bin width 3°. The vertical axis represents number of fast-Ca-transients for the bins of the histogram. The horizontal axis represents the phase in terms of angular degree. Lower middle panels (c): Circular diagrams constructed for the same set of data showing mean resultant vectors. The colored lines represent the mean resultant vectors for the Ca-transients recorded from each of the ROIs and the arrows represent the mean resultant vector for the Ca-transients recorded from all the ROIs (see Methods). Bottom panels (d): Statistical values for all the fast-Ca-transients recorded from all the ROIs; frequency of the Ca-transients, total number of the transients (N), mean phase (), circular standard deviation in terms of angular degree (acSD), mean resultant length (
), and the significance probability for the null hypothesis of uniform circular distribution (P).
Fig 8.
Dependence of the extent of entrainment and the relative change in frequency on the field strength.
(A) Blue: The mean resultant length plotted against field intensity (FI). The relation was fitted to an exponential function
= 0.13+0.65 (1-exp(-0.45FI)), (r = 0.92). Red: The circular standard deviation (cSD = sqrt(-2log(
)) plotted against field intensity. The relation was fitted to an exponential function cSD = 0.72 + 16.81×exp(-(FI +9.27)2/5.862), (r = 0.89). 5 slices, 24 trials (4 Hz, 19 trials; 2 Hz, 4 trials; 1 Hz, 1 trial). (B) Red: The circular standard deviation in terms of angular degree (acSD = (180°/π)cSD) plotted against field intensity. The relation was fitted to an exponential function acSD = 40.76 + 1.13×105×exp(-(FI +26.01)2/9.652), (r = 0.89). Green: The linear standard deviation (lSD) plotted against field intensity. The relation was fitted to an exponential function lSD = 48.83 + 2.48×103×exp(-(FI +15.43)2/-8.362),(r = 0.74). Note that there was no noticeable change in the extent of entrainment within the intensity range of 5–22 mV/mm. (C) The mean phase
plotted against field intensity. Marks of the same color represent data from the same slice preparation. Filled marks are used for the five cases in which electric field with the same intensity and frequency were applied. Note that the mean phase was different from one trial to the next. (D) The relative change in frequency of fast-Ca-transients during field stimulation plotted against field intensity. Orange: Relative change in frequency (RCF) during a preferred hemi-circle, that is a hemi-circle with 180° angular width with the center angle pointing toward the mean phase (RCF = 0.60FI+1, r2 = 0.46). Blue: Relative change in frequency during anti-preferred hemi-circle, the opposite hemi-circle of the field (RCF = exp(-0.13FI), r2 = -0.52). Red: The ratio of the relative change in frequency (RRCF) during preferred and anti-preferred hemi-circle (RRCF = 3.30FI+1, r2 = 0.46). Black: The mean relative change in frequency (mRCF) during full circle compared to the frequency in condition with no field stimulation (mRCF = 0.27FI+1, r2 = 0.13). 5 slices. 22 trials (4 Hz, 17 trials; 2 Hz, 4 trials; 1 Hz, 1 trial).